Abstract: Dysregulation of protein kinase activity has been implicated in a number of diseases, including cancer, diabetes and chronic inflammation. Therefore, protein kinases have emerged as one of the most important drug targets in modern drug discovery. These efforts have resulted in the development of a vast array of small molecule inhibitors that interact with the ATP-binding sites of protein kinases and are able to block their catalytic activities. Interestingly, many of these pharmacological agents exploit the conformational flexibility of the ATP-binding sites of protein kinases by binding to different active site conformations. For example, the drugs Gleevec (imatinib) and Nexavar (sorafenib) bind to an ATP-binding site conformation of their kinase targets that involves displacement of a catalytically important structural element called the DFG motif. While it has been speculated that certain modes of kinase inhibition lead to more desirable clinical outcomes, there have not been any systematic comparisons at the biochemical and/or cellular level. Our hypothesis is that ATP-competitive inhibitors that stabilize different ATP-binding site conformations of multi-domain protein kinases will have divergent effects on domains and interactions outside of the active site. This leads to the exciting possibility that it may be possible to obtain different phenotypic responses through the inhibition of the same kinase active site by varying the mode of ATP-binding site occupancy. To test this hypothesis, we propose to study how different modes of inhibition affect the SRC family of kinases (SFKs), which are well-characterized, multi- domain tyrosine kinases. SFKs play diverse roles in multiple signaling processes and are important therapeutic targets. Furthermore, it has been demonstrated that beyond their catalytic activities, SFKs also have a number of non-catalytic scaffolding functions that are dependent on their regulatory domains.